CWI1-2

YY1-induced upregulation of FOXP4-AS1 and FOXP4 promote the proliferation of esophageal squamous cell carcinoma cells

Yonghui Li1, Tingting Li1, Yongbin Yang2, Wenli Kang3, Shaoyong Dong1, Shujie Cheng4, *

Abstract

Esophageal squamous cell carcinoma (ESCC) belongs to one of the most common malignant tumors worldwide and possesses high mortality. Long non-coding RNAs (lncRNAs) have been demonstrated to be essential biological participants in the progression of ESCC. Based on bio-informatics prediction, forkhead box P4 antisense RNA 1 (FOXP4-AS1) and forkhead box P4 (FOXP4) were upregulated in esophageal carcinoma samples and were positively correlated with each other. Present study aimed to explore the function of FOXP4-AS1 and FOXP4 in ESCC cells. Function assays disclosed that knockdown of FOXP4-AS1 or FOXP4 efficiently suppressed cell proliferation and induced cell apoptosis. Moreover, FOXP4-AS1 positively regulated FOXP4 by interacting with insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) to stabilize FOXP4 mRNA. Additionally, FOXP4-AS1 could upregulate the expression of FOXP4 by sponging miR-3184-5p. Finally, we found that YY1 is a transcription factor that can transcriptionally activate both FOXP4-AS1 and FOXP4 in ESCC cells. In a word, YY1-induced upregulation of FOXP4-AS1 and FOXP4 promote the proliferation of ESCC cells

Key words: ESCC; FOXP4-AS1; FOXP4; IGF2BP2; proliferation

1. Introduction

Esophageal cancer (EC) ranks the third in digestive system malignancies and ranks the seventh in all commonest cancers (Bray et al., 2018; Siegel et al., 2014). Majority of EC cases are defined as esophageal squamous cell carcinoma (ESCC) (Torre et al., 2015; Yang, Sun, et al., 2018). Although traditional therapies are effective for ESCC patients in early stage, the prognosis for advanced patients is still unfavorable (He et al., 2014; Herskovic et al., 2012; Smyth et al., 2017). Therefore, exploring novel molecular mechanism underlying the progression of ESCC is urgently necessary.
The protein-coding gene accounts for 2% in human genome and most of transcripts are noncoding RNAs (ncRNAs) (Ling et al., 2015). Long non-coding RNAs, as the members of ncRNAs, exerted a noticeable function in regulating cancer progression. LncRNAs are a class of 200 nucleotides (nt) without protein-coding ability (Marchese et al., 2017). LncRNAs can alter gene expression through multiple regulatory approaches, such as chromatin modification, transcriptional regulation and post-transcriptional regulation (Chen et al., 2018; Harrap, 2016; Jalali et al., 2015; Laham-Karam et al., 2017). Aberrant expression of lncRNAs has been proved to be associated with abnormal biological behaviors of malignant tumors, such as cervical cancer, hepatocellular carcinoma, glioma and breast cancer (Aalijahan & Ghorbian, 2019; Gao et al., 2017; Kong et al., 2017; Luan et al., 2017). Besides, lncRNAs can regulate the expression of their nearby genes to regulate cancer progression (Su et al., 2017; Wang et al., 2018; Zhu et al., 2016). Despite that lncRNAs play a role in many cellular progressions, such as cell proliferation, apoptosis, migration, invasion, and so on (Sun et al., 2013), the biological functions and molecular mechanisms of lncRNAs in human cancers has not been well researched.
In this research, based on GEPIA database, we found that lncRNA forkhead box P4 antisense RNA 1 (FOXP4-AS1) and its nearby gene forkhead box P4 (FOXP4) are both upregulated in EC samples and positively correlated with each other. Previously, FOXP4-AS1 has been reported to be an oncogene in colorectal cancer (Li et al., 2017) and osteosarcoma (Yang, Ge, et al., 2018). Cancer facilitator role of FOXP4 was researched in breast cancer (Ma & Zhang, 2019), prostate cancer (Huang et al., 2019) and renal carcinoma (Xiong et al., 2019). However, the role and molecular mechanism of FOXP4-AS1 and FOXP4 in ESCC are still unclear. Therefore, this study aimed to investigate the function and mechanism of FOXP4-AS1 and FOXP4 in ESCC progression. We designed and conducted loss-of function assays to demonstrate the effects of FOXP4-AS1 or FOXP4 on ESCC cell proliferation and apoptosis.
Mechanistically, we found the localization of FOXP4-AS1 in ESCC cells. Bioinformatics prediction and mechanism test were utilized to prove the interaction between FOXP4-AS1 and FOXP4 in ESCC cells. Post-transcriptional regulatory effect of FOXP4-AS1 on FOXP4 was explored and analyzed. Finally, we searched out a transcription factor YY1 which could transcriptionally activate FOXP4-AS1 and FOXP4. Taken together, our study revealed a novel molecular pathway in cellular process of ESCC.

2. Materials and methods

2.1 Cell lines and culture

Human esophageal epithelial cell line (HEEC) and five acknowledged human ESCC cell lines (EC9706, EC109, KYSE410, KYSE150 and KYSE450) were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were routinely cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) containing penicillin-streptomycin and 10% fetal bovine serum (FBS; Invitrogen, MA, USA). Cells were grown in an incubator at 37˚C with 5% CO2.

2.2 RNA isolation and qRT-PCR

Total cellular RNA was extracted using TRIzol reagent RNA extraction kit (Molecular Research Center, Cincinnati, OH, USA). miRcute miRNA isolation kit (Tiangen) was applied to extract miRNAs. The first cDNA strand was generated using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). miScript Reverse Transcription kit (Qiagen) was used for reverse transcription of cDNAs. Amplification of cDNAs was made by using SYBR1 Premix Ex Taq™ II (Takara). Real-time PCR detection of genes was performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Ct values of genes were normalized to GAPDH or U6. Results were expressed by the comparative CT method (2−ΔΔCt). All procedures were run in triplicate. Related primers were listed in Supplementary Table 1.

2.3 Cell transfection

KYSE150 and KYSE450 cells were planted into culture plates until reached 70-80% confluence. The short hairpin RNAs (shRNAs, GeneCopoecia, Guangzhou, China) against human FOXP4-AS1 (sh-FOXP4-AS1), FOXP4 (sh-FOXP4) and YY1 (sh-YY1) and negative control shRNA (sh-NC) were separately transfected into cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the standard method. The negative control shRNAs were synchronously synthesized by GeneCopoecia. The miR-3184-5p mimics (mimics) and miR-NC were purchased from GenePharma (Shanghai, China). Overexpression vector of IGF2BP2 or YY1 was achieved by cloning the whole sequence of IGF2BP2 or YY1 (pcDNA-IGF2BP2 or pcDNA-YY1) into pcDNA 3.1 vector (GeneCopoecia). Empty vector was used as the negative control (NC). Forty-eight hours later, cells were reaped for functional or mechanism assays.

2.4 Cell proliferation assays

The transfected KYSE150 and KYSE450 cells were seeded in 96-well plates and diluted at a density of 1 × 104 cells/ml. 10 μL of Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was put into each well and incubated with cells for 2 hours at 37°C. The absorbance at 450 nm was measured by a microplate reader at 24, 48, 72 and 96 h. For EdU assay, EdU incorporation assay kit was bought from Ribobio (Guangzhou China). KYSE150 and KYSE450 cells were cultured with 100 μL of 50 μM EdU medium diluent for 3 hours. Following fixation in 4% paraformaldehyde, cells were treated with ® 488 fluorescent staining reaction liquid at 37°C 100 μL of 1 × Apollofor half an hour. Cell nuclei was subjected to DAPI staining in the dark. Experimental data were expressed as the ratio of EdU positive cells to DAPI positive cells. Cell proliferation assays were carried out at least three times.

2.5 Caspase-3 activity detection

The caspase-3 activity was detected by Caspase-3 Activity Kit (Abcam, Cambridge, MA, USA). Total protein extracted from cells was planted into 96-well plates, followed by incubation with reaction buffer and caspase-3 substrate at 37°C for 4 hours. Caspase-3 activity was assessed using a microplate reader (Tecan Group Ltd., Männerdorf, Switzerland) at 405 nm. This assay was conducted in triplicate.

2.6 Western blot assay

Protein samples (40 μg) isolated from cells were diluted and electrophoresed in 12% SDS-PAGE. Followed by transferring onto polyvinylidene difluoride (PVDF) membranes, 5% skimmed milk was utilized to block the membranes at room temperature for two hours. Primary antibodies, including anti-Bax (Abcam, Cambridge, MA, USA; ab32503), anti-Bcl-2 (Abcam, ab32124), anti-caspase-3 (ab13847), anti-cleaved caspase-3 (ab2302), anti-PARP (ab74290), anti-cleaved PARP (ab32064), anti-FOXP4 (Abcam, ab119404) and anti-GAPDH (Abcam, ab157156) were diluted at 1:1000 using the Primary Antibody Dilution Buffer (Beyotime, Guangzhou, China) and cultured with cells at 4oC all night. Afterwards, membranes were washed in Tris-buffered saline (TBST) and incubated with the corresponding secondary antibody against rabbit IgG conjugated to horseradish peroxidase (Abcam, 1:5000). An enhanced chemiluminescence (ECL) detection system (Bio-Rad, Hercules, CA, USA) was applied to visualize protein bands. GAPDH was seen as the internal control.

2.7 Subcellular fractionation assay

The PARIS™ Kit (Invitrogen) was applied to carry out subcellular fractionation assay in accordance with recommendation provided by supplier. KYSE150 and KYSE450 cells in cell fractionation buffer were subjected to centrifugation. The supernatant was collected. The remaining lysates were rinsed in cell fractionation buffer. Cell disruption buffer was utilized to lyse cell nuclei. Thereafter, the lysate was cultured with the supernatant, 2 × lysis / binding solution and ethanol. At length, cytoplasmic and nuclear RNAs were eluted and examined by qRT-PCR. Biological triplicates were carried out.

2.8 Fluorescence in situ hybridization (FISH)

The probe for FOXP4-AS1-FISH was designed and synthesized by Ribobio Company (Guangzhou, China). GAPDH and U6 were used as the cytoplasmic and nuclear positive control, respectively. KYSE150 and KYSE450 cells were placed on culture slides and fixed in 4% paraformaldehyde, following sealing with pre-hybridization buffer at 37°C for 4 hours. FISH probe was added into the hybridization mixture all night. Afterwards, slides were washed in washing buffer containing saline-sodium citrate (SSC). Cells were stained with DAPI for visualizing nuclei. Lastly, cells were observed under an Olympus fluorescence microscope (IX73; Olympus Corp., Tokyo, Japan). Experiments were performed for three times for biological replicates.

2.9 RNA immunoprecipitation (RIP) assay

The Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was utilized for RIP assay. 1 × 107 KYSE150 and KYSE450 cells were incubated in RIP lysis buffer. The lysates were co-immunoprecipitated with antibody against IGF2BP2 (Abcam). qRT-PCR was used to analyze the recovered RNAs. The total RNA (Input control) and normal IgG control were simultaneously subjected to qRT-PCR to verify the detected signal from the RNA specially binding to IGF2BP2. To confirm the combination of FOXP4-AS1, miR-3184-5p and FOXP4, the lysates were cultured with protein A/G sepharose beads conjugated to antibodies against Ago2 (Millipore, Massachusetts, USA) or IgG (Millipore). Each experimental procedure was repeated for three times.

2.10 Chromatin immunoprecipitation (ChIP) assay

The SimpleChIP® Enzymatic Chromatin IP Kit (CST, Danvers, MA, USA) was applied to ChIP assay. KYSE150 and KYSE450 cells were subjected to formaldehyde for 10 min to obtain DNA-protein cross-links. The lysates were sonicated into 200-bp – 500-bp chromatin fragments, followed by immunoprecipitation with 2 μg of YY1-specific antibody (Abcam) and 2 μg of IgG as control overnight at 4oC. 30 μL magnetic beads were incubated with the complex for 2 h at 4°C. Lastly, the precipitated chromatin was washed, purified and analyzed by qRT-PCR.

2.11 Luciferase reporter assay

The wild type sequence of FOXP4-AS1 or FOXP4 containing the binding sites with miR-3184-5p was cloned into the firefly luciferase gene in pmirGLO luciferase vector (GeneChem, Shanghai, China) and was named as FOXP4-AS1-WT or FOXP4-WT. The mutant form of FOXP4-AS1 or FOXP4 3’ UTR (FOXP4-AS1-MUT or FOXP4-MUT) were established using the GeneTailor™ Site-Directed Mutagenesis System (Invitrogen). KYSE150 and KYSE450 cells in24-well plates were separately co-transfected with the aforementioned reporter plasmids and miR-3184-5p mimics or miR-NC using Lipofectamine2000. For FOXP4-AS1 or FOXP4 promoter luciferase analysis, FOXP4-AS1 or FOXP4 promoter containing YY1 binding sites was cloned into pGL3-Basic reporter vector (Promega, Madison, WI, USA). Cells were co-transfected with indicated reporter plasmids and pcDNA-YY1 or NC. At last, luciferase activities were evaluated by Dual-Luciferase Reporter Assay System (Promega Corporation, Fitchburg, WI, USA), normalizing to Renilla luciferase activity.

2.12 Bioinformatics analysis

The expression patterns of FOXP4-AS1 and FOXP4 in EC along with the expression correlation between them in ESCC were downloaded from GEPIA database (http://gepia.cancer-pku.cn/index.html). MiRNAs containing the binding sites with FOXP4-AS1 and FOXP4 were predicted using DIANA TOOLS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=lncBa se/index) and starBase v3.0 (http://starbase.sysu.edu.cn/). Potential transcription regulators for FOXP4-AS1 and FOXP4 were searched out from UCSC (http://genome.ucsc.edu/). The DNA motif of YY1 and putative binding sequences of YY1 in FOXP4-AS1 or FOXP4 promoter were obtained from JASPAR (http://jaspar.genereg.net/).

2.13 Colony formation assay

were inoculated into 6-well (Sigma-Aldrich). At last, the visible colonies ( ≥ 50 cells) were manually counted.

2.14 Statistical analysis

All experimental results were acquired from at least three different biological replications. Data were presented as the mean ± standard deviation (SD). The significance of difference was assessed by Student’s t-test and one-way ANOVA using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The threshold of p value was set as 0.05 to be statistically significant.

3. Results

3.1 Upregulation of FOXP4-AS1 and FOXP4 promoted ESCC cell proliferation and induced cell apoptosis

Based on GEPIA data, lncRNA FOXP4-AS1 and its nearby gene FOXP4 were both upregulated in esophageal carcinoma samples (Figure 1A). The positive expression association between them in esophageal carcinoma samples was revealed (Figure 1B). Moreover, lncRNA FOXP4-AS1 (Figure 1C, left) and FOXP4 mRNA ((Figure 1C, right) were expressed at a relative higher level in ESCC cell lines compared to the normal cell line HEEC. KYSE150 and KYSE450 exhibited highest expression level of FOXP4-AS1 and FOXP4. Therefore, we silenced FOXP4-AS1 (Figure 1D, left) and FOXP4 (Figure 1D, right) in KYSE150 and KYSE450 cells by using specific shRNAs. Functionally, knockdown of FOXP4-AS1 and FOXP4 efficiently suppressed cell proliferation (Figure 1E-F, S1A). According to the results of apoptosis test, caspase-3 activity, protein level of Bax, cleaved capsase-3, cleaved PARP were increased in cells transfected with sh-FOXP4-AS1 or sh-FOXP4, while the protein level of Bcl-2 was decreased (Figure 1G-H), indicating the promoting effects of silenced FOXP4-AS1 or FOXP4 on cell apoptosis. These findings suggested that FOXP4-AS1 and FOXP4 might contribute to ESCC progression.

3.2 FOXP4-AS1 interacted with IGF2BP2 protein to enhance the stability of FOXP4 mRNA

To determine the regulatory pattern of FOXP4-AS1 in ESCC, we detected the subcellular localization of FOXP4-AS1 in two ESCC cells. As presented in Figure 2A and Figure 2B, FOXP4-AS1 was predominantly located in the cytoplasm of ESCC cells, indicating the post-transcriptional regulation of FOXP4-AS1. Moreover, the mRNA and protein level of FOXP4 were decreased in cells transfected with sh-FOXP4-AS1 (Figure 2C). Therefore, we made mechanism investigation to explore the underlying regulation pattern of
FOXP4-AS1 on FOXP4. Through bioinformatics analysis, we found that IGF2BP2 was a RBP (RNA-binding protein) that might simultaneously bind to FOXP4-AS1 and FOXP4. RIP assay demonstrated that FOXP4-AS1 could interact with IGF2BP2 (Figure 2D). Importantly, the mRNA expression level of FOXP4 was decreased by the knockdown of IGF2BP2 (Figure 2E).
Mechanistically, lncRNAs can upregulate mRNAs by binding to RBPs to stabilize mRNAs (Lan et al., 2018; Zhang et al., 2018). In this regard, we conducted RIP assay to validate the binding of IGF2BP2 to FOXP4 (Figure 2F). Moreover, the binding of IGF2BP2 to FOXP4 was attenuated by the knockdown of FOXP4-AS1 (Figure 2G). Finally, we detected the effect of IGF2BP2 and FOXP4-AS1 on the mRNA stability of FOXP4 by treating ESCC cells with Actinomycin D (an antagonist of mRNA stability). It was found that the stability of FOXP4 was decreased by sh-FOXP4-AS1, but was recovered by the overexpression of IGF2BP2 (Figure 2H). Based on these data, we confirmed that lncRNA FOXP4-AS1 could upregulate FOXP4 by interacting with IGF2BP2 to stabilize FOXP4 mRNA.

3.3 FOXP4-AS1 acted as a sponge of miR-3184-5p to upregulate FOXP4

LncRNAs can post-transcriptionally regulate miRNAs to release the downstream mRNAs (Cao et al., 2016; Lü et al., 2015). On this basis, we explored whether FOXP4-AS1 positively regulated FOXP4 by acting as a miRNA sponge. According to the search results of starBase and DIANA, three putative miRNAs could simultaneously bind with FOXP4-AS1 and FOXP4 (Figure 3A). Then, we examined the expression change of these three miRNAs in cells transfected with FOXP4-AS1. As illustrated in Figure 3B, miR-3184-5p exhibited the highest fold change in response to the knockdown of FOXP4-AS1 (Figure 3B). The binding sequence between them was obtained by using bioinformatics prediction tools (Figure 3C). Luciferase reporter assay was further conducted to verify the interaction between FOXP4-AS1/FOXP4 and miR-3184-5p. The experimental results showed that the luciferase activity of reporters containing the wild type FOXP4-AS1 or FOXP4 was significantly decreased by miR-3184-5p mimics (Figure 3D). However, no obvious changes were observed in the luciferase activity of mutant reporters. Ago2-RIP assay further demonstrated that FOXP4-AS1, miR-3184-5p and FOXP4 (Figure 3E) were abundantly enriched in Ago2-precipitated RISC (RNA induced silence complex). Moreover, the mRNA and protein level of FOXP4 were all decreased in two ESCC cells transfected with miR-3184-5p mimics compared to NC group (Figure 3F). These results indicated that FOXP4-AS1 positively regulated FOXP4 in ESCC cells by sponging miR-3184-5p.

3.4 YY1 transcriptionally activated FOXP4-AS1 and FOXP4 in ESCC cells

Seventy-eight potential transcription regulators for FOXP4-AS1 and FOXP4 were searched out from UCSC. Then, we overexpressed all these 78 potential regulators to examine their influences on the expression level of FOXP4-AS1 and FOXP4. According to the PCR results, FOXP4-AS1 and FOXP4 had the most significant response to the overexpression of YY1 (Figure 4A). In addition, silencing of YY1 led to the downregulation of FOXP4-AS1 and FOXP4 in ESCC cell lines (Figure 4B). The impact of YY1 on the transcription of FOXP4-AS1 and FOXP4 was further detected. We obtained the DNA motif of YY1 from UCSC (Figure 4C). Using bioinformatics tool, we predicted one binding site of YY1 on FOXP4-AS1 promoter and two binding sites of YY1 on FOXP4 promoter. According to luciferase activity analysis, the sequence from -1341 to -1352 bp was responsible for the interaction between YY1 and FOXP4-AS1 promoter, and sequence from -507 to -518 bp was responsible for the interaction between YY1 and FOXP4 promoter (Figure 4D-E). The affinity of YY1 to FOXP4-AS1 and FOXP4 promoter was further verified by ChIP assay (Figure 4F). Collectively, we determined that YY1 transcriptionally activated both FOXP4-AS1 and FOXP4.

4. Discussion

It is acknowledged that 90% of human genomes are transcribed into non-protein-coding RNA (Tehrani et al., 2018). LncRNAs control cellular functions by interacting with chromatin-modifying complex or transcriptionally/post-transcriptionally regulating gene expression (Schmitt & Chang, 2016). FOXP4-AS1 has been reported to exert cancer-promoting function in cancers, for illustration, FOXP4-AS1 is involved in the initiation and progression of osteosarcoma through suppressing the expression of LATS1 by binding to LSD1/EZH2 protein (Yang, Ge, et al., 2018). LncRNAs have been demonstrated to be crucial regulators of their nearby genes. In our present study, we found that FOXP4-AS1 and FOXP4 were two upregulated genes in EC samples. Besides, their carcinogenesis has been reported in previous studies, for example, FOXP4-AS1 is closely associated with dismal prognosis and facilitates cell proliferation and inhibits cell apoptosis in colorectal cancer (Li et al., 2017). LncRNA LOC105372579 contributes to proliferation and EMT process in hepatocellular carcinoma by modulating miR-4316/FOXP4 axis (E et al., 2019). Overexpression of miR-491-5p restrains cell proliferation and stimulates cell apoptosis through targeting FOXP4 in human osteosarcoma (Yin et al., 2017). Further, the expression correlation between FOXP4-AS1 and FOXP4 was proved to be positive. According to the results of functional assays, knockdown of FOXP4-AS1 and FOXP4 led to the inhibited cell proliferation and increased cell apoptosis. These data indicated that FOXP4-AS1 and FOXP4 exerted oncogenic roles in ESCC progression, which conformed to previous researching results.
In the present study, we found that FOXP4-AS1 positively regulated the mRNA and protein level of FOXP4. Thus, we investigated the mechanism which contributed to the regulation of FOXP4-AS1 on FOXP4. In this research, through bioinformatics analysis and RIP assay, we determined that IGF2BP2 could bind to FOXP4-AS1 and FOXP4. Furthermore, the binding of IGF2BP2 to FOXP4 was attenuated by the silence of FOXP4-AS1. Finally, based on previous studies that lncRNAs could modulate mRNA stability (He et al., 2018; Xiong et al., 2017), we proved that the mRNA stability of FOXP4 was decreased by the knockdown of FOXP4-AS1 and was rescued by the introduction of IGF2BP2. To sum up, FOXP4-AS1 upregulated the expression of FOXP4 and enhanced the mRNA stability of FOXP4 by cooperating with IGF2BP2 in ESCC cells.
Cytoplasmic localization of FOXP4-AS1 indicated that FOXP4-AS1 could post-transcriptionally regulate gene expression. At post-transcriptional level, lncRNAs can serve as ceRNAs to upregulate mRNAs by sponging miRNAs (Lv et al., 2016). ceRNA is an important way for lncRNAs to exert their functions in various cancers. For instance, lncRNA HULC promotes liver cancer via upregulating HMGA2 expression through sequestrating miR-186 (Wang et al., 2017). LncRNA SNHG1 accelerates the development of non-small cell lung cancer via overexpressing MTDH and sponging with miR-145-5p (Lu et al., 2018). LncRNA XIST promotes colorectal cancer progression by modulating miR-137/EZH2 pathway (Liu et al., 2018). On this basis, we confirmed that FOXP4-AS1 acted as a ceRNA in ESCC cells to upregulate FOXP4 via sponging miR-3184-5p.
Transcriptional regulation is an important mechanism that results in the upregulation of lncRNAs and mRNAs (Cui et al., 2015; Sun et al., 2018). There into, transcription factor plays a noticeable role. For example, LINC01048 is activated by USF1 and motivates cell proliferation in cutaneous squamous cell carcinoma via regulating TAF15/YAP1 (Chen et al., 2019). In our present study, we found that YY1 was a putative transcription regulator for both FOXP4-AS1 and FOXP4. Mechanism experiments demonstrated that YY1 promoted the transcription of FOXP4-AS1 and FOXP4 by binding to their promoter region in ESCC cells.
Collectively, our current study demonstrated that upregulation of FOXP4-AS1 promoted cell proliferation and induced cell apoptosis in ESCC cells by interacting with IGF2BP2 protein and sequestering miR-3184-5p to up-regulate FOXP4. This study revealed a novel molecular pathway in ESCC, which may contribute to enrich the study on the molecular mechanism associated with ESCC progression. The shortcoming for this study is that we only focused on cell proliferation and apoptosis in ESCC cells. Hence, we aimed to examine more cell functions including cell migration, invasion and EMT process in future studies. Moreover, we would explore the clinical significance of FOXP4-AS1 and FOXP4 in our future investigation.

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